Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus

The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish

Retraction: Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

Besnier Jean-Michel. Lucien Brunelle (1923-1995). In: Raison présente, n°114, 2e trimestre 1995. Foucault et le projet critique. p. 1

AST2 interacts with <i>Pl</i>RACK1.

<p>A) <i>Pl</i>RACK1 was identified as an AST2 binding protein using a far western assay with recombinant GST-AST2 or GST as a control. A HPT protein extract was subjected to SDS-PAGE, electroblotted to a PVDF membrane and overlayed with either GST (left) or GST-AST2 (right) alone and binding was detected using a GST antibody. B) The binding between recombinant AST2 with <i>Pl</i>RACK1 was confirmed by a GST pull-down assay using GST-AST2 as the binding protein and proteins in a HPT cell lysate as bait. GST was used as a control in both assays. C) GST pull-down of His-Trx-AST2 by GST-<i>Pl</i>RACK1. The bound proteins were analyzed by 12.5% SDS-PAGE. Bands corresponding to GST and GST-<i>Pl</i>RACK1 were detected with an anti-GST antibody (lanes 1–4). The eluted material was also examined for the presence of AST2 with an anti-His antibody (lanes 5–8). Lanes 9 and 10 contain purified His-Trx-AST2 and HisTrx, respectively.</p

Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

<div><p>Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish <i>Pacifastacus leniusculus</i> is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.</p> </div

A 400-kDa CLK-<i>Pl</i>BMAL1 complex is present in the light in the crayfish brain.

<p>A) Immunoprecipitation (IP) of endogenous CLOCK (CLK) and <i>Pl</i>BMAL1 in the brain (B) and HPT. Total proteins were extracted from the brain and HPT and were immunoprecipitated (IP) with the antibodies (Ab) indicated on the top of the blots, followed by western blot (WB) detection with antibodies against CLK or BMAL1 as shown at the bottoms of the blots. Reducing and non-reducing conditions of the samples are indicated by +DTT or −DTT, respectively. B) The levels of the CLK-<i>Pl</i>BMAL1 and AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The AST2-<i>Pl</i>RACK1 heterodimer and CLK were detected by western blotting using antibodies against AST2 and CLK, respectively. Time points were taken at 03:00, 06:00, 12:00, 18:00, and 21:00 (n = 4). An actin protein was used as an internal control. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). C) Relative amounts of CLK-<i>Pl</i>BMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. D) Relative amounts of AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. E) Relative amounts of AST2-<i>Pl</i>RACK1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. F) Relative amounts of CLK protein in brain extracts at different time points (n = 3) as determined by western blotting. Average protein level in Graphs C–F was quantitated using Quantity One. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. G) Melatonin injection inhibited the formation of the CLK-<i>Pl</i>BMAL1 complex; this inhibition is mediated by the AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 complex. The levels of the CLK-<i>Pl</i>BMAL1 and AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The level of β-actin was used as an internal control. H) Relative levels of CLK-<i>Pl</i>BMAL1 and AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 complexes <i>in vivo</i>, in the brain of crayfish after injection of melatonin and then brain extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). I) Relative levels of CLK-<i>Pl</i>BMAL1 and AST2-<i>Pl</i>RACK1-<i>Pl</i>BMAL1 complexes <i>in vivo</i>, in the HPT of crayfish after injection of melatonin and then HPT extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). The level of β-actin was used as an internal control. Asterisks indicate significant differences (*<i>P</i><0.05, **<i>P</i><0.01). Quantity One analysis was used to quantify the intensity of protein bands. Graphs (H and I) represent the quantification of each complex formation, using Quantity one. Results are representative of three independent experiments. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).</p

AST2 expression is induced by melatonin treatment <i>in vitro</i> and <i>in vivo</i>.

<p>A) Relative expression of AST1 mRNA estimated by qPCR, after incubation with melatonin in cultured HPT cells <i>in vitro</i> at daytime. Black bars = melatonin (3 µM), white bars = control. B) Relative expression of AST2 mRNA estimated by qPCR, after incubation with melatonin in cultured HPT cells <i>in vitro</i> at daytime. Black bars = melatonin (3 µM), white bars = control. C) Relative expression of AST1 mRNA in hemocytes estimated by qPCR, after injection of melatonin in live crayfish. Black bars = melatonin (4.3 nmol/g), white bars = control. D) Relative expression of AST2 mRNA in hemocytes estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. E) Relative expression of AST1 mRNA in HPT estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. F) Relative expression of AST2 mRNA in HPT estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. G) Relative expression of AST1 mRNA in the brain estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. H) Relative expression of AST2 mRNA in the brain estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. Expression of the 40S ribosomal protein was used as an internal control. Error bars indicate standard deviation (SD) from three replicates and the experiment has been repeated three times with similar results. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test.</p

Melatonin induces higher AST2 protein levels <i>in vitro</i> and <i>in vivo</i>.

<p>A) Relative levels of AST1 or AST2 protein in cultured HPT cells <i>in vitro</i> as estimated by ELISA, after incubation with melatonin. Black bars = melatonin (3 µM), white bars = control. The level of β-actin was used as an internal control. B) Relative levels of AST1 in the HPT, brain and hemocytes in live crayfish as estimated by ELISA, after injection of melatonin. Black bars = melatonin (4.3 nmol/g), white bars = control. The level of β-actin was used as an internal control. C) Relative levels of AST2 in the HPT, brain and hemocytes in live crayfish as estimated by ELISA, after injection of melatonin. Black bars = melatonin (4.3 nmol/g), white bars = control. The level of β-actin was used as an internal control. The asterisks indicate significant differences (*P<0.05); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. D) Western blot analysis of AST2 in the brain and HPT at 9:00 and 20:00, using an antibody against AST2. The expression level of actin was used as an internal control. Light and dark periods are indicated on the top of the blot.</p

<i>Pl</i>RACK1, <i>Pl</i>BMAL1, and AST2 form an approximately 200-kDa protein complex during the dark period.

<p>A) Relative amounts of AST2, <i>Pl</i>RACK1 and <i>Pl</i>BMAL1 proteins in brain extracts as determined by ELISA at 3 h after light turned off or on. B) Relative amounts of AST2, <i>Pl</i>RACK1 and <i>Pl</i>BMAL1 proteins in HPT extracts as determined by ELISA at 3 h after light turned off or on. The asterisks indicate significant differences (*P<0.05); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. C) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed that a <i>Pl</i>RACK1-AST2 complex was present in the brain (B) during the day. D) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed the presence of a high-molecular-weight complex (approximately 200 kDa) composed of all three proteins in the brain (B) at night. C–D) The immunoprecipitated complex was analyzed by western blotting (WB) using another antibody. “B” and “HPT” represent brain and hematopoietic tissue, respectively. The antibodies used for immunoprecipitation (IP) and detection (WB) is indicated at the top and bottom of the blots, respectively, and +DTT and −DTT represent reducing and non-reducing conditions, respectively. Molecular masses are indicated at the left.</p

<i>Pl</i>RACK1 can bind to <i>Pl</i>BMAL1.

<p>A) <i>In vitro</i> GST pull-down assay of His-Trx-<i>Pl</i>BMAL1 by GST-<i>Pl</i>RACK1. The elution fractions of the GST-<i>Pl</i>RACK1 pull-down assay were examined by western blot analysis using anti-GST and anti-His antibodies. Lanes 1 and 5: elution fraction of GST-<i>Pl</i>RACK1 pull-down of HisTrx-<i>Pl</i>BMAL1; Lanes 2 and 6: GST-<i>Pl</i>RACK1 pull-down of His-Trx; Lanes 3 and 7: GST pull-down of HisTrx-<i>Pl</i>BMAL1; Lanes 4 and 8; GST pull-down of His-Trx. B) The protein-protein interaction of <i>Pl</i>RACK1 and <i>Pl</i>BMAL1 was analyzed by far western blotting. C) Binding of AST2 and <i>Pl</i>BMAL1 to GST-<i>Pl</i>RACK1. GST-<i>Pl</i>RACK1 (0, 5, 50 or 500 ng) was bound to Glutathione Sepharose beads and incubated with 500 ng His-Trx-<i>Pl</i>BMAL1 and 500 ng His-Trx-AST2. Bound proteins were eluted and immunoblotted for <i>Pl</i>BMAL1 and AST2 using an anti-His antibody and for <i>Pl</i>RACK1 with an anti-GST antibody.</p